Plasmid DNAs are potential therapeutic agents for many human and animal diseases. Many of these plasmid DNAs are too large to produce by chemical synthesis and therefore are most efficiently produced by propagating them in a host cell, which is in turn grown on a nutrient medium to high density. The DNA is subsequently recovered from the host cells. Isolation and purification of plasmid DNA from complex mixtures involves multiple stages of purification. The first stage includes concentrating the cells from fermentation media, which is composed of unconsumed nutrients as well as cellular waste products. The next stage requires lysis or disruption of the cells, which results in a complex biochemical mixture of cellular constituents including the plasmid DNA of interest. This is typically done in the presence of chaotropic salts to minimize enzymatic activity, which can degrade plasmid DNA, or can be accomplished by mechanical means such as with a pressure cell, sonicator, hydrodynamic shearing device, or bead beater in conjunction with an enzyme inhibitor to minimize DNA degradation. The next stage of plasmid preparation involves physically separating soluble plasmid DNA from insoluble material such as membranes, denatured proteins and large chromosomal DNA fragments by centrifugation, differential precipitation or filtration. This produces a cleared lysate containing plasmid DNA, which can be further purified by direct precipitation, phase partitioning or by adsorption to various resins via ion exchange or hydrophobic interaction methods and combinations thereof, which may be followed by precipitation or phase partitioning of plasmid DNA. Chromatographic purification can be accomplished by batch or column chromatographic methods and may involve indirect, via compatible ion, or direct adsorption methods. All these methods are well known to those skilled in the art and each step is designed to remove impurities and contaminants from plasmid DNA. All purification regimens can be improved by reducing the amount and complexity of the material from which the plasmid DNA is isolated. This includes potential contaminants in the media as well as material produced by the host cell.
One significant impurity present in cellular preparations derived from bacterial cells is endotoxin, which is in part derived from lipopolysaccharide (LPS) present in the outer membrane of bacteria. Gram-negative bacteria such as E. coli require at least a minimal structural component of LPS for viability and wholesale deletion of genes that encode the pathways required for biosynthesis of these structures is lethal. However, a few genes involved in LPS synthesis are not essential, and the cell can tolerate deletion or loss of function of these specific genes. Many attempts have been made to manipulate such genes to improve the endotoxin profile of bacteria without demonstrable success. One notable exception is the discovery that deletion of msbB, which encodes myristoyl acyltransferase, produces a cell with a 1000-10,000-fold reduction in the ability to stimulate TNFα by human tissue culture cells (Sommerville et al., J. Clin. Invest. 1996 97:359). The use of msbB mutants to produce LPS deficient cells capable of producing DNA with lowered endotoxin levels and use of such cells as tumor targeted vectors is disclosed in a number of U.S. patents (e.g., U.S. Pat. Nos. 6,548,287; 6,080,849; and 5,997,881).
The chromosome of E. coli contains many cryptic prophage and insertion sequence (IS) elements that represent horizontally transferred genes that have moved into E. coli over time. Removal of these horizontally transferred sequences and other non-essential genes produces multiple deletion strain (MDS) variants such as that described by Blattner and co-workers (Posfai et al., Science 2006 312:1044; U.S. Pat. No. 6,989,265 and in PCT/US03/01800, published as WO 03/070880, all of which are incorporated herein by reference in their entirety). These cells are unique in that they lack significant numbers of genes relative to other E. coli strains commonly used in production of plasmid DNA. However, these strains remain completely prototrophic and are capable of robust growth on defined minimal media. Defined minimal media for E. coli typically includes the following (g/l): 5 g glucose, 6 g Na2HPO4, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl. 0.12 g MgSO4 and 0.01 g CaCl2.
Plasmid DNA preparations from cells grown in defined minimal media will lack many of the undefined components entrained from the undefined components of rich media necessary for efficient growth of auxotrophic bacterial cell lines commonly used to produce plasmid DNA. The use of defined minimal media, with a limited number of chemically known nutrient sources, for growth of plasmid containing bacterial cells will result in lowering the number of media derived contaminants, as well as provide a more uniform cellular composition from which plasmid DNA must be subsequently purified. In addition, the lack of uncontrolled lysis during fermentation of MDS strains due to removal of all cryptic prophage lysis functions, as well as the reduced biochemical complexity of MDS cells due to removal of many genes from the chromosome, also reduces the number of proteins and other biological components within the lysate. Indeed, plasmid DNA produced from MDS strains which remain completely proficient for LPS production and retain all genes known to affect LPS synthesis, have significantly reduced levels of endotoxin by LAL assay relative to other commonly used bacterial cell lines, even when grown in rich undefined media.
Production of plasmid DNAs for use as human therapeutics requires minimizing the amount of bacterial endotoxin remaining in the purified DNA product. Biological production of plasmid DNAs in bacterial hosts provides an efficient and scalable method for producing large quantities of plasmids. However, purification of plasmid DNA from bacterial hosts, by any combination of methods currently practiced does not completely eliminate endotoxin from the prepared DNA. If plasmid DNA could be produced having lower levels of endotoxin than available by current methods, a significant advance in the art would result.